Application of North American Ginseng Polysaccharide in Prevention and Treatment of Respiratory Syncytial Virus

Abstract

This invention discloses the application of *Adenophora stricta* polysaccharide in the prevention and treatment of respiratory syncytial virus (RSV), belonging to the field of biomedical technology. This invention is the first to discover that *Adenophora stricta* polysaccharide can inhibit RSV and can serve as a specific active ingredient for RSV prevention and treatment. The research results of this invention overcome the technical blind spot where the overall efficacy of traditional Chinese medicine components cannot be attributed to specific components, precisely identifying that *Adenophora stricta* polysaccharide alone can exert anti-RSV effects, providing a new target for designing RSV inhibitors and developing combination drug strategies covering multiple respiratory viruses.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of North American ginseng polysaccharide in the prevention and treatment of respiratory syncytial virus. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Respiratory syncytial virus (RSV), since its first identification in 1957, has become the most common pathogen causing respiratory diseases worldwide. Almost all adults have experienced RSV infection during childhood, sometimes even experiencing recurrent infections. Primary RSV infection typically leads to lower respiratory tract bronchiolitis and pneumonia. Severe RSV infection is characterized by cough, wheezing, shortness of breath, dyspnea, chest wall retraction, and cyanosis. Globally, more than 3 million people are hospitalized annually due to RSV infection, and approximately 60,000 die, posing a serious threat to human health. Currently, there are no effective prevention and treatment methods for RSV infection in China; treatment mainly focuses on supportive care to relieve symptoms. Commonly used drugs include interferon, corticosteroids, and bronchodilators. However, long-term use can lead to drug resistance, hormone resistance, or dependence, inducing related complications. Therefore, developing effective, safe, and long-term suitable new drugs is of great significance.

[0004] North American ginseng is the root of *Glehnia littoralis*, a plant in the Apiaceae family. It is rich in diverse components, including polysaccharides, volatile oils, coumarins, amino acids, and trace elements. The *Compendium of Materia Medica* records that *Glehnia littoralis* is sweet, bland, and cold in nature, light and delicate, specifically tonifying lung qi, thus benefiting the spleen and kidneys. Therefore, it is suitable for those whose metal element is susceptible to fire. It clears lung heat and treats chronic cough and pulmonary atrophy. North American ginseng is sweet, slightly bitter, and slightly cold in nature; it enters the lung and stomach meridians, possessing the effects of nourishing yin and moistening the lungs, benefiting the stomach and generating fluids. It is a core drug for treating yin deficiency and qi and yin deficiency syndromes, and is often used clinically to treat symptoms such as dry cough due to lung heat, hemoptysis due to overwork, stomach yin deficiency, heat-related injury to body fluids, and dry throat and thirst. North American ginseng polysaccharide is a plant polysaccharide extracted from North American ginseng, possessing antioxidant and hypoglycemic functions. Currently, there are no reports of using North American ginseng polysaccharide for the prevention and treatment of RSV virus infection. Summary of the Invention

[0005] In view of the above-mentioned prior art, the purpose of this invention is to provide the application of North American ginseng polysaccharide in the prevention and treatment of respiratory syncytial virus (RSV), which solves the problems of lack of specific drugs, large side effects of Western medicine, and unclear active ingredients of traditional Chinese medicine in the existing treatment of RSV, in order to provide a reference for the research and development and clinical application of anti-RSV drugs.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides the use of North American ginseng polysaccharide in the preparation of medicaments for the prevention and / or treatment of respiratory syncytial virus infection.

[0008] The preparation method of the North American ginseng polysaccharide is as follows:

[0009] (1) Degreasing: Take dried North American ginseng root slices, crush and sieve them, add petroleum ether, heat and reflux at 60°C for 1-2 hours, filter and collect the residue, dry at room temperature to remove solvent and obtain North American ginseng powder;

[0010] (2) Water extraction: Add distilled water to the North American ginseng powder obtained in step (1), heat to boiling for 1-2 h, filter and collect the filtrate, and concentrate by rotary evaporation at 60℃ to obtain concentrated liquid;

[0011] (3) Protein removal: Mix the protein removal reagent and the concentrated solution obtained in step (2) at a volume ratio of 1:4, vortex for 8-10 min, sonicate at 200 W for 2-3 min, centrifuge and collect the supernatant to obtain the crude polysaccharide extract of Glehnia littoralis.

[0012] (4) Alcohol precipitation: Add anhydrous ethanol to the crude polysaccharide extract of Glehnia littoralis obtained in step (3) until the final concentration of ethanol is 80% (v / v). After the reaction is complete, collect the precipitate by centrifugation and freeze-dry it to obtain Glehnia littoralis polysaccharide.

[0013] Preferably, in step (2), the ratio of the North American ginseng powder to distilled water is 1:10.

[0014] Preferably, in step (3), the protein impurity removal reagent is a mixture of chloroform and n-butanol in a volume ratio of 4:1.

[0015] In a second aspect, the present invention provides a drug for preventing and treating respiratory syncytial virus infection, wherein the drug uses the above-mentioned North American ginseng polysaccharide as an active ingredient.

[0016] The drug also includes pharmaceutically acceptable solvents or excipients.

[0017] Preferably, the dosage form of the drug is capsule, tablet, powder, pill, suppository, injection, or granule.

[0018] The beneficial effects of this invention are:

[0019] This invention is the first to discover that polysaccharides from *Adenophora stricta* can inhibit respiratory syncytial virus (RSV), thus serving as a specific active ingredient for RSV prevention and treatment. The findings overcome the technical limitation that the overall efficacy of traditional Chinese medicine components cannot be attributed to specific ingredients, precisely identifying that *Adenophora stricta* polysaccharides alone can exert anti-RSV effects. This provides a new target for designing RSV inhibitors and developing combination therapy strategies covering multiple respiratory viruses. Attached Figure Description

[0020] Figure 1 Image showing the appearance of polysaccharides from Glehnia littoralis.

[0021] Figure 2 The graph shows the determination of polysaccharide content in Glehnia littoralis.

[0022] Figure 3 The image shows the infrared spectrum of polysaccharides from Glehnia littoralis.

[0023] Figure 4 The diagram shows the monosaccharide composition determination of polysaccharides from *Adenophora stricta* and a comparison diagram of standard products. Figure 4 Figure A shows the comparison chart of the standard sample; Figure 4 Figure B shows the monosaccharide composition determination of polysaccharides from Glehnia littoralis.

[0024] Figure 5 The image shows the surface morphology of polysaccharides from *Adenophora stricta*. Figure 5 Image A shows the surface morphology of North American ginseng polysaccharides at a magnification of 500. Figure 5 Image B shows the surface morphology of North American ginseng polysaccharide at a magnification of 2000. Figure 5 Image C shows the surface morphology of North American ginseng polysaccharide at a magnification of 10000.

[0025] Figure 6 To detect cell viability using CCK8.

[0026] Figure 7 This is a diagram of cellular pathological changes.

[0027] Figure 8 This refers to the RSV virus titer.

[0028] Figure 9 The expression level of RSV-F mRNA was detected by qRT-PCR. Detailed Implementation

[0029] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0030] The specific embodiments of the present invention will be described in further detail below with reference to examples. The following detailed descriptions are illustrative and intended to provide further explanation of this application, rather than limiting the scope of the invention.

[0031] The RSV virus used in this embodiment of the invention was provided by the Institute of Basic Medical Sciences, Shandong Academy of Medical Sciences, passaged in the P2 laboratory of Shandong Academy of Traditional Chinese Medicine, and stored in a -80℃ refrigerator.

[0032] This RSV virus was documented in Wang X, Ren W, Wang P, et al. Investigating the active components and mechanistic effects of Forsythia suspensa Leaf against RSV via the PI3K / Akt-NLRP3 pathway. Heliyon . 2024;10(19):e38285. Published 2024 Sep21.

[0033] Example 1: Preparation of North American Ginseng Polysaccharide

[0034] (1) Degreasing: Take 100g of dried North American ginseng root slices (produced in Laiyang, Shandong Province, purchased from Shandong Baiweitang Chinese Medicine Slices Co., Ltd.), crush and sieve them, add 400ml of petroleum ether, heat and reflux at 60℃ for 2h, filter and collect the residue, dry at room temperature to remove the solvent and obtain North American ginseng powder.

[0035] (2) Water extraction: Add distilled water to the North American ginseng powder at a material-to-liquid ratio of 1:10, heat to boiling for 1 hour, filter and collect the filtrate, repeat the extraction 3 times, combine the filtrates, and concentrate by rotary evaporation at 60°C to obtain about 200 ml of concentrated liquid.

[0036] (3) Protein removal: The Sevage method was used to remove protein impurities. A protein removal reagent was prepared by mixing chloroform and n-butanol at a volume ratio of 4:1. The protein removal reagent and the concentrated solution were mixed at a volume ratio of 1:4 and vortexed for 10 min. To improve the protein removal efficiency, the mixture was sonicated at 200 W for 2 min and centrifuged at 3000 g for 10 min. The supernatant was collected to obtain the crude polysaccharide extract of Glehnia littoralis.

[0037] (4) Alcohol precipitation: Add anhydrous ethanol to the crude polysaccharide extract of Glehnia littoralis at a volume ratio of 1:4 until the final ethanol concentration is 80% (v / v). After sufficient precipitation, centrifuge at 3000g for 10min, collect the precipitate, and freeze-dry it to obtain Glehnia littoralis polysaccharide. Figure 1 ).

[0038] Example 2: Determination of polysaccharide content and structural characterization of Glehnia littoralis

[0039] (1) Determination of polysaccharide content in Glehnia littoralis by phenol-sulfuric acid method:

[0040] A standard curve was prepared by diluting the glucose standard stock solution. The content of polysaccharides in Glehnia littoralis was measured using the phenol-sulfuric acid method, and the absorbance value at 490 nm was measured using a full-wavelength microplate reader.

[0041] glucose standard curve as follows Figure 2 Plotting glucose content as the x-axis and absorbance as the y-axis, the glucose standard curve was obtained as Y = 0.008353*X + 0.09823, with R² = 0.9995. Substituting these values ​​into the curve, the crude polysaccharide content of *Adenophora stricta* was calculated to be 82.32%.

[0042] The functional group types of the prepared North American ginseng polysaccharide were determined by spectrometer.

[0043] The results were obtained by scanning and analysis using a Nicolet iZ-10 Fourier transform infrared spectrometer. Figure 3 As shown, 3211.97cm -1 The significant signal is the absorption peak of the stretching vibration of OH, a characteristic peak of carbohydrates. (2924.49 cm⁻¹) -1 The absorption peak at 1012.9 cm⁻¹ is attributed to the CH stretching vibration, which is consistent with the origin of methylene and methine groups on saturated alkyl chains or sugar rings; furthermore, the absorption peak at 1012.9 cm⁻¹... -1 There is an absorption peak at this point, which is attributed to the stretching vibration of CO and is direct evidence of the cyclic structure and linkages of carbohydrates.

[0044] (3) The monosaccharide composition of the prepared North American ginseng polysaccharide was determined by mass spectrometry.

[0045] Depend on Figure 4 It is known that the polysaccharide of North American ginseng is a heteropolysaccharide composed of glucose (Glc), galactose (Gal), arabinose (Ara), galacturonic acid (Gal-UA), and rhamnose (Rha), with molar percentages of 90.64%, 3.43%, 3.00%, 2.28%, and 0.65%, respectively. Among them, glucose is the main monosaccharide component.

[0046] (4) The surface morphology of the prepared North American ginseng polysaccharide was examined by scanning electron microscopy.

[0047] The surface morphology of North American ginseng polysaccharide was obtained using scanning electron microscopy, such as... Figure 5As shown, at a magnification of 500, the polysaccharide from *Adenophora stricta* exhibits an amorphous sheet-like structure. The higher glucose content may lead to increased interchain hydrogen bonds, resulting in a highly folded, loosely sheet-like microstructure that facilitates polysaccharide dissolution. Under even higher magnification, the polysaccharide displays a network-like porous structure, and irregular granular structures are observed adhering to the surface of the polysaccharide sample, indicating its excellent adsorption capacity.

[0048] Example 3: CCK8 assay for the cytotoxicity of North American ginseng polysaccharides

[0049] Human laryngeal epidermoid carcinoma cells Hep-2 (provided by the Institute of Basic Medical Sciences, Shandong Academy of Medical Sciences, passaged in the P2 laboratory of Shandong Academy of Traditional Chinese Medicine, and stored at -80℃) were seeded into 96-well cell culture plates at 100 μL / well, incubated at 37℃ for 24 h with 5% CO2. The culture medium was then discarded, and the concentrations of *Adenophora stricta* polysaccharide were diluted with 2% FBS DMEM medium to 12.5, 25, 50, 100, 200, 400, 800, and 1600 μg / mL. A control group of normal cells receiving only maintenance medium (2% FBS DMEM) was included. After 48 h of culture, CCK8 solution was added to each well at a ratio of 10:1, and after incubation for 2 h, the absorbance was measured using a microplate reader at an OD value of 450 nm.

[0050] Result: Passed Figure 6 It can be seen that different concentrations of North American ginseng polysaccharide have no cytotoxicity on Hep-2 cells.

[0051] Example 4: Inhibitory effect of North American ginseng polysaccharide on RSV virus

[0052] (1) Observation of the degree of cytopathic effect

[0053] Hep-2 cells were seeded in 96-well plates and cultured at 37°C with 5% CO2 for 24 h. After the culture medium was discarded, 12.5, 50, 200, and 800 μg / mL of North American ginseng polysaccharide were added to each well and cultured for 2 h. After washing with PBS and discarding the supernatant, 100 μL of 100 TCID45 solution was added to each well. 50 After incubating RSV virus for 2 hours, the supernatant was discarded and washed with PBS. Then, 12.5, 50, 200 and 800 μg / mL of North American ginseng polysaccharide were added respectively. Cells without polysaccharide and virus treatment were used as blank control groups (mock). After culturing for 24 hours, the cytopathic effects were observed and recorded under a microscope.

[0054] Result: Passed Figure 7 It can be seen that, compared with the blank control group, the cytopathic effect was alleviated after intervention with different concentrations of North American ginseng polysaccharide. When the concentration was 200 and 800 μg / mL, it could exert a significant anti-RSV virus effect.

[0055] (2) Virus TCID 50 Measurement

[0056] Hep-2 cells were seeded in 6-well plates and cultured at 37°C with 5% CO2 for 24 h. The culture medium was discarded, and North American ginseng polysaccharide at concentrations of 12.5, 50, 200, and 800 μg / mL was added to each well for 2 h. After washing with PBS and discarding the supernatant, 1 ml of 100 TCID45 solution was added to each well. 50 After RSV virus incubation for 2 hours, the supernatant was discarded and the cells were washed with PBS. Then, North American ginseng polysaccharide at concentrations of 12.5, 50, 200, and 800 μg / ml was added, respectively. Cells without polysaccharide or virus treatment served as a blank control group (mock). After 24 hours of culture, the supernatant was collected and serially diluted 10-fold to 10⁻⁶. -8 TCID of the virus was determined using the Karber method. 50 .

[0057] from Figure 8 It can be seen that, compared with the blank control group, when the concentration of North American ginseng polysaccharide was 50, 200, and 800 μg / mL, the RSV virus titer of Hep-2 cells was significantly reduced after intervention with North American ginseng polysaccharide, indicating that North American ginseng polysaccharide has an inhibitory effect on RSV, and the inhibitory effect increases with the increase of North American ginseng concentration.

[0058] (3) Detection of RSV-F mRNA expression level

[0059] The F gene (NC-001803.1) of respiratory syncytial virus (RSV) encodes a key surface glycoprotein of the virus and plays a central role in the RSV infection mechanism, especially in the irreplaceable role in viral invasion of host cells, mediating membrane fusion and immune escape.

[0060] Hep-2 cells were seeded in 6-well plates and cultured at 37°C with 5% CO2 for 24 h. The culture medium was discarded, and North American ginseng polysaccharide at concentrations of 12.5, 50, 200, and 800 μg / mL was added to each well for 2 h. After washing with PBS and discarding the supernatant, 1 ml of 100 TCID45 solution was added to each well. 50After incubating RSV virus for 2 hours, the supernatant was discarded and the cells were washed with PBS. Then, 12.5, 50, 200, and 800 μg / ml of *Adenophora stricta* polysaccharide were added, respectively. Cells without polysaccharide and virus treatment served as a mock control. After 24 hours of culture, RNA was extracted from the cells using the Vazyme FastPure Complex Tissue / Cell Total RNA Isolation Kit (RC113). The RNA was reverse transcribed into cDNA using the HiScriptIII RT SuperMix for qPCR (+gDNA wiper) (R323) kit. qRT-PCR was performed using the primers shown in SEQ ID NO.1-SEQ ID NO.2 with the ChamQ Universal SYBR qPCRMaster Mix (Q711) kit to detect the relative expression level of RSV-F mRNA.

[0061] F (SEQ ID NO.1):TTGGATCTGCAATCGCCA;

[0062] R (SEQ ID NO. 2): CTTTTGATCTTGTTCACTTCTCCTTCT.

[0063] from Figure 9 It can be seen that, compared with the blank control group, when the concentration of North American ginseng polysaccharide was 50, 200, and 800 μg / mL, the expression level of RSV virus F gene mRNA in Hep-2 cells was significantly reduced after intervention with North American ginseng polysaccharide, indicating that North American ginseng polysaccharide has an inhibitory effect on RSV, and the inhibitory effect increases with the increase of North American ginseng concentration.

[0064] The above description is merely a preferred embodiment of this application and is not intended to limit the application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications made within the spirit and principles of this application are not permitted.

[0065] Equivalent substitutions and improvements should all be included within the scope of protection of this application.

Claims

1. The application of North American ginseng polysaccharide in the preparation of drugs for the prevention and / or treatment of respiratory syncytial virus infection; The preparation method of the North American ginseng polysaccharide is as follows: (1) Degreasing: Take dried North American ginseng root slices, crush and sieve them, add petroleum ether, heat and reflux at 60°C for 1-2 hours, filter and collect the residue, dry at room temperature to remove solvent and obtain North American ginseng powder; (2) Water extraction: Add distilled water to the North American ginseng powder obtained in step (1), heat to boiling for 1-2 h, filter and collect the filtrate, and concentrate by rotary evaporation at 60℃ to obtain concentrated liquid; (3) Protein removal: Mix the protein removal reagent and the concentrated solution obtained in step (2) at a volume ratio of 1:4, vortex for 8-10 min, sonicate at 200 W for 2-3 min, centrifuge and collect the supernatant to obtain the crude polysaccharide extract of Glehnia littoralis. (4) Alcohol precipitation: Add anhydrous ethanol to the crude polysaccharide extract of Glehnia littoralis obtained in step (3) until the final concentration of ethanol is 80%. After the reaction is complete, centrifuge to collect the precipitate and freeze-dry it to obtain Glehnia littoralis polysaccharide. In step (2), the ratio of the North American ginseng powder to distilled water is 1:10; In step (3), the protein impurity removal reagent is a mixture of chloroform and n-butanol in a volume ratio of 4:

1.

2. The application according to claim 1, characterized in that, The drug also includes pharmaceutically acceptable solvents or excipients.

3. The application according to claim 1, characterized in that, The dosage form of the drug is capsule, tablet, powder, pill, suppository, injection, or granule.

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